Cumitech 24: Rapid Detection of Viruses by Immunofluorescence

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CUMITCCH

Cumitech IA Blood Cultures II June 1982 Cumitech 2A Laboratory Diagnosis of Urinary Tract Infections March 1987 Cumitech 3 Practical Quality Control Procedures for the Clinical Microbiology Laboratory September 1976 Cumitech 4 Laboratory Diagnosis of Gonorrhea October 1976 Cumitech 5 Practical Anaerobic Bacteriology April 1977 Cumitech 6 New Developments in Antimicrobial Agent Susceptibility Testing September 1977 Cumitech 7A Laboratory Diagnosis of Lower Respiratory Tract Infections September 1987 Cumitech 8 Detection of Microbial Antigens by Counterimmunoelectrophoresis December 1978 Cumitech 9 Collection and Processing of Bacteriological Specimens August 1979 Cumltech IO Laboratory Diagnosis of Upper Respiratory Tract Infections December 1979 Cumitech I I Practical Methods of Culture and Identification of Fungi in the Clinical Microbiology Laboratory August 1980 Cumitech I2 Laboratory Diagnosis of Bacterial Diarrhea October 1980 Cumitech I3 Laboratory Diagnosis of Ocular Infections 9 May 1981 Cumitech I4 Laboratory Diagnosis of Central Nervous System Infections January 1982 Cumitech I5 Laboratory Diagnosis of Viral Infections March 1982 Cumitech I6 Laboratory Diagnosis of the Mycobacterioses March 1983 Cumitech I7 Laboratory Diagnosis of Female Genital Tract Infections August 1983 Cumitech 18 Laboratory Diagnosis of Hepatitis Viruses January 1984 Cumitech I9 Laboratory Diagnosis of Chlamydial and Mycoplasmal Infections August 1984 Cumitech 20 Therapeutic Drug Monitoring: Antimicrobial Agents October 1984 Cumitech 21 Laboratory Diagnosis of Viral Respiratory Disease March 1986 Cumitech 22 Immunoserology of Staphylococcal Disease August 1987 Cumitech 23 Infections of the Skin and Subcutaneous Tissues June 1988 l

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Cumifechs should be cited as follows, e.g.: Minnich, 24, Rapid detection of viruses by immunofluorescence. for Microbiology, Washington, DC.

Editorial Board for ASM Cumitechs: Steven Mar-tone, John E. McGowan, Jr., Glenn D. Roberts, and Ake S. Welssfeld.

L. L., T. F. Smith, and C. G. Ray. 1988. Cumitech Coordinating ed., S. Specter. American Society

C Specter, Charman, Carl Abramson, Wllllam J. James W. Smith, John A. Smith, Thomas J. Tlnghltella,

The purpose of the Cumitech series is to provide consensus recommendations of-the-art operating procedures for clinical microbiology laboratories which routine or new methods. be procedures gwen are not proposed as “standard” methods.

Coovrlght

0 1988 American Society 1913 I St, NW Washtngton. DC 20006

by the authors as to appropriate statemay lack the facilities for fully evaluating

for MIcrobIology

RAPID DETECTION OF VIRUSES BY IMMUNOFLUORESCENCE LINDA L. MINNICH, Arizona 85724 THOMAS

Departments of Pathology and Pedia tries, Arizona Health Sciences Center, Tucson,

F. SMITH, Section of Clinical Microbiology,

Mayo Clinic, Rochester, Minnesota 55905

C. GEORGE RAY, Departments of Pathology and Pediatrics, Arizona Health Sciences Center, Tucson, Arizona 85724 COORDINATING

STEVEN SPECTER, Department Tampa, Florida 33612

of Medical

EDITOR

Microbiology

and Immunology,

University of South Florida,

secondary antiserumis then conjugated with the fluorescent dye. This has the advantage of decreasingthe number of antisera which must be labeled, but it increasesthe risk for nonspecific cross-reactivity. In theory, the indirect system should provide an amplification of the reaction and may increase sensitivity over the direct system. However, avidity of the primary antiserum is also a factor, and increased sensitivity may not be observed with use of the indirect system. The complement-mediatedindirect immunofluorescence system (also known as the anticomplementary immunofluorescence system) is limited by the ability of the primary antiserum to fix complement. However, it may be necessaryfor the detection of small amounts of antigen or use of low-avidity antibody. After the primary antigen-antibody reaction a source of complement, often guineapig serum, is added to the reaction. A fluorescent dye-conjugated antiserum to the complement protein(s) is then added in a third incubation step. The two incubation and wash steps involved in the indirect method and the three incubation and wash steps involved in the complement-mediated indirect immunofluorescencemethods increase the time and labor required for routine use. The additional antiserum requirements increase the potential for reactions with other antigensto which any one of the animals used for antibody production might have been exposed. Selection of methodsis dependent on the amount of antigen present, the availability of conjugated antisera, and the desire of the laboratorian.

This Cumitech reviews the selection, collection, transport, and processingof specimensfor diagnosisof viral infections by immunofluorescenceeither directly in clinical samplesor in cell cultures, including the recently developed shell vial assay methods. The basic methodology of immunofluorescence,microscopesemployed in immunofluorescence procedures, quality control, resolution of technical problems, and interpretation of results are reviewed. Immunofluorescencehas been applied to the detection of parasitic, fungal, bacterial, viral, and other antigens. This has been possiblebecausethe biological activity of the antibody is not affected by covalent binding with a fluorescent dye. The limitations of immunofluorescence are imposed only by the presence of detectable antigen in a specimenand the ability to produce specific antibody to react with it.

PRINCIPLES OF PROCEDURES There are three basic methodsfor the detection of antigensby immunofluorescence:direct, indirect, and complement-mediatedindirect systems. In the direct method, the antibody to a specific antigen(s) is conjugated directly with a fluorescent dye (Fig. 1A). The initial work of labeling each antigen-specific (primary) antiserum is greatest for this system, but as there is only one incubation and wash step, subsequent testing is the least time-consuming. The necessity of labeling each primary antiserum is avoided by the use of either the indirect (Fig. 1B) or the complement-mediatedindirect (Fig. 1C) system. The indirect system involves the MICROSCOPES AND LIGHT SOURCES production of the primary antisera in a limited Use of any of the above systems requires a number of animal species, such as guinea pigs, mice, rabbits, goats, and cows. The secondary special light source which produces the approantiserumis produced to the immunoglobulin of priate excitation light in the wavelength range the speciesusedfor the primary antiserum. This that produces maximum emissionof the fluores1

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FIG. 1. Major methods for the performance of immunofluorescence testing. (A) Direct; (B) indirect; anticomplement indirect. Symbols: 0, complement; F, labeled antibody; A, unlabeled antibody.

cent dye used for conjugation. A filter system must then be used to screen out the excitation light from the emitted fluorescent light and precisely localize the specific immunologic interaction. Mercury arc or halogen lamps are frequently used as light sources. Mercury arc lampswill often yield increasedsensitivity of the system and allow use of more than one fluorescent dye with the appropriate barrier systems. Disadvantages of mercury arc lamps are the expenseof original purchase, the limited number of hours they can be used, and the necessity of maintaining a log of use hours. This log is required because the amount of emitted light produced by the lamp gradually decreasesover time. Alternatively, a timer may be used to measure lamp use hours. Halogen lamps are much less expensive to purchase initially, last longer, and simply burn out, therefore not requiring a usagelog. Selection of the systemto be used is determined by funds, desired use, and convenience. Although either light source is satisfactory for the detection of viral antigens, it is important to titrate the antisera and conjugateswith the samemicroscopeand light source to be usedfor routine studiesto yield consistent results. Once the choice of light source has been made, the next decision is to select either transmitted light or epifluorescence. Most modern fluorescence microscopes employ epifluorescence, in which oil is only required with the use of oil immersion lenses.Use of epifluorescence microscopy also has increasedthe sensitivity of the immunofluorescencesystems. REAGENTS AND SUPPLIES The labelingof antibody may be accomplished with many fluorescent dyes. Fluoresceinisothiocyanate (FITC) and rhodamine are the most commonly used dyes. FITC may be used with either a halogen or a mercury arc light source. Rhodaminemay be usedonly with a mercury arc light source. Different filter systemsare require*!

24

(C)

for the use of the two dyes, but this fact also allows dual labeling, to facilitate simultaneous detection of two antigensin the samespecimen. Antisera may be either polyclonal or monoclonal. Polyclonal antisera typically recognize multiple antigensand are commonly produced in rabbits, guineapigs, mice, rats, hamsters,goats, and cows. A disadvantageis that the animal also may have produced antibody to related or unrelated antigens that may be found in clinical specimens.Occasionally it is difficult to produce antisera with high titers or high avidity to a specific virus becauseof failure of the animal to respond to those antigens. This may sometimes be overcome by the useof adjuvants or selection of another speciesfor immunization. Monoclonal antibodies have less potential for crossreactivity, but present the additional problem of occasionally being too selective for routine clinical use. For example, a monoclonal antibody may be highly specific in the detection of one gene product, but may not effectively detect all strains of a particular viral serotype. This problem of high selectivity has usually been overcome either by pooling two or more monoclonal antibodies or by selecting a monoclonal antibody which reacts to an antigen shared by all strains within the serotype (20, 23, 25, 26, 29, 37). The selection of direct rather than indirect and of polyclonal rather than monoclonal antibodies is largely determined by the availability of commercial reagents.At present, reagentsfor the detection of herpes simplex virus (HSV) and respiratory syncytial virus (RSV) are usually readily available for both the direct and the indirect methods. Reagentsfor the detection of parainfluenza virus, influenza virus, and adenoviruses are becoming more readily and consistently available. Careful in-house evaluation of all commercial reagentsis advised (30). Once the critical decisionshave been made as to the choice of microscope, the system to be developed (direct, indirect, or complement-mediated indirect), the dye to be used, and the

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VIRUS DETECTION

polyclonal or monoclonal antisera, other seemingly mundane factors which can significantly influence the successor failure of using immunofluorescencefor viral antigen detection must be evaluated. Although ordinary glass slides may be used, the cost of reagents usually precludes this. Two types of slidesmay be used to avoid the waste of reagents. The first is ringed slidesmadeby painting circles on ordinary glass slides. If done by a laboratorian, a pipe of the appropriate diameter may be dipped into an oil-basedenamelpaint and applied to the slide. Ringed slidesmay be purchased from a variety of sourcesand are available in a variety of ring sizes and number of rings per slide. Slides with the entire slide painted, leaving only the wells clear, are also available. More recently, Tefloncoated slides have become commercially available in a variety of well sizes, configurations, and colors. Theseoffer the distinct advantage of providing a hydrophobic barrier between the wells, thus reducing the risk of inadvertent mixing of antisera during the incubation process. These slidesalso markedly decreasethe amount of antiserumusedper specimen.Prepared slides are valuable, since compared with the cost for reagents,the cost of prepared slidesaddslittle to overall expense. Many specimensfor the detection of viral antigens are collected with swabs which are subsequently placed in viral transport media. Type 1 cotton- or Dacron-tipped swabs with aluminum shafts are recommendedfor the collection of nasopharyngeal (NP) and urethral specimens.Calcium alginate swabs should not be used for the collection of specimensfor viral studiessincethey disintegratein the fluid transport media and inhibit some viruses in culture detection systems. The aluminum shaft is more flexible than the stainless-steelshaft and therefore more comfortable for the patients, as well as easier to break or cut off into the vial of transport media. Cotton- or Dacron-tipped applicators with plastic or wooden shafts may be used for the collection of specimensfrom the throat, cervix, vagina, etc., or cutaneous and mucous membranelesions. While salinemay be usedfor washingcells or slidesafter incubation with antisera, phosphatebuffered saline (PBS) is more frequently selected. This decreasesthe chance of problems due to an excessively high or low pH resulting from the contamination of glasswareor water. It can be relatively inexpensive and easy to prepare PBS with a pH of 7.3 to 7.8 by making a 10x solution which is diluted in the appropriate volume of distilled water as needed. Mounting fluid may be purchased from a variety of commercial sources or inexpensively made by adding approximately one part 10x

BY IMMUNOFLUORESCENCE

3

Tris buffer to nine parts glycerol to obtain a pH of 8.0 to 9.5. Viscosity of the buffered glycerol may be adjusted with the addition of Tris buffer as long as the pH range is maintained. Immersion oil is too acid and quenches fluorescence rapidly; therefore, it should never be applied directly to the stained specimen. Number 1 cover slips are used by many laboratorians to avoid problems with light refraction and focusing due to the thickness of the cover slip; no. 1% and 2 cover slips are not usually satisfactory for immunofluorescencework becauseof their increasedthickness. Conjugatesand antisera are best stored frozen (-20°C or colder) in the concentrated form, with dilution at the time of use. Stability is maintained in the frozen state for at least several years. However, storage should not be in a self-defrostingfreezer becauseof the deleterious effects of repeated freeze-thaw cycles. Diluted antisera are usually stableat 2 to 8°C for several months. It is desirableto divide the antisera and conjugated antisera into smallportions for longterm frozen storage. QUALITY

CONTROL

Quality control of immunofluorescence includes the evaluation of sensitivity and specificity of the reagents. It involves their titration with heterologousantigenswhich may be found in the system. Reactivity with uninfected cell cultures and microorganisms which may be found as contaminantsin clinical samplesshould also be ascertained. Preliminary

Evaluation

of Antisera

The initial quality control of the direct system is simplest. Serial dilutions of the conjugated antisera are overlaid onto slidesprepared from cell cultures infected with the homologousvirus. Initial dilutions may vary from 1:2 to 1:500, depending upon the concentration of the antisera. Some commercialreagentsare provided at a working dilution. Incubation times vary from 15 to 60 min, depending upon the temperature and antisera. Incubations may be performed at room temperature or 35 to 37”C, but must be done in a humidified chamber to avoid drying of the slides.Humidified chambersmay be devised by placing a wet paper towel in the bottom of a disposablepetri dish or plastic chamber. Wash times may vary from two 10-swashesfor monoclonal antisera to three 5-min washesfor polyclonal antisera. Selected incubation and wash times are observed. The smearsare then evaluated for specific fluorescencewith minimal background. At this optimal dilution the antiserum is tested for cross-reactivity with heterologous viruses that may be found in clinical samples, using infected cell cultures or, when available,

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actual clinical samples. For example, a conjugate to HSV should be evaluated for crossreactivity against varicella-zoster virus (VZV) and cytomegalovirus (CMV). Similarly, a conjugated antiserum for a respiratory agent should ideally be evaluated for reactivity against influenza virus type A (both HlNl and H3N2 strains), influenza virus type B, RSV, parainfluenza virus types 1, 2, and 3, adenovirus, poliovirus types 1, 2, and 3, and, if possible, measles and mumps. All conjugated antisera should be evaluated for reactivity against the cell culture systems in use in the laboratory. In addition, the conjugated antiserum should be evaluated for reactivity with normal tissue (e.g., lung and brain) if it is to be used for the detection of viral antigens in biopsies. Clinical specimens known to be positive for the homologous virus as well as heterologous viruses are ideal, but may not be available on initial evaluation or in small laboratories. The indirect system requires a cross titration. Unless provided at a working dilution, the primary antiserum is serially diluted, usually starting with a 15 or 1:lO dilution, and the FITClabeled secondary antiserum is used at 1: 10 for the initial evaluation. Once the titer of the primary antiserum is determined (the highest dilution to produce bright fluorescence, with dimming of fluorescence in the subsequent dilutions), the optimal dilution of the primary antiserum is used and the conjugated secondary antiserum is serially diluted. Thus, the optimal dilutions of the primary and conjugated secondary antisera may be determined with two procedures, and minimal amounts of reagents are used with microdilution methods. Tips or pipettes must be changed after each dilution is made to assure accurate, reproducible titers. This is especially important with monoclonal antibodies. The complement-mediated indirect system requires three titrations. First, the primary antiserum is titrated with the complement diluted 1: 10 and the FITC-conjugated anticomplement antiserum diluted 1: 10. Second, with the primary antiserum at an optimal dilution, the complement is titrated, usually using eight serial twofold dilutions starting at 1: 10. Third, with the primary antiserum and complement at optimal dilutions, the FITC-conjugated anticomplement antiserum is titrated. The reagents of either the indirect or the complement-mediated indirect system are then checked at the working dilutions for cross-reactivity, as for the direct system.

CUMITECH

24

control must be addressed.Appropriate quality control is determined by the experience of the laboratory and the criteria and standardsestablishedby the applicableregulatory and certifying agencies. In theory, running a positive control each use-dayservesto assurethe quality control of the reagents, including conjugates, buffers, and the technique. It should be noted that the use of prepared slides of infected cell culture does not control for specimen collection and preparation. In addition, the pattern of immunofluorescence and amount of antigen in an infected cell culture may markedly differ from what is actually encountered in clinical specimens. Therefore, the infected cell culture control could indicate that the system is within limits, when in fact it may not be capable of detecting the viral antigen in clinical specimens. To minimize these problems, infected cell cultures should usually be harvested for the preparation of positive control slides before the appearance of cytopathic effect. Conversely, slides prepared from uninfected cell cultures may show a negative result when the test system produces falsely positive reactivity in clinical samples.Once an antiserum or conjugatehasbeen subjectedto a rigorous initial evaluation with standardization, daily negative controls are usually of little practical value. While infected and uninfectedcell culture slides may fulfill criteria for regulatory agenciesand are often the only controls indicatedby manufacturing companies,they do not replace the more important needfor a careful initial evaluation of individual reagentlots. It is of utmost importanceto have the specific methodsof quality control stated in the written proceduresand to carefully document the quality control performed in order to satisfy laboratory standards and accrediting agencies. Written records should include the antiserumor conjugatetype, lot numbers,date in service, date last used, initial quality control results, and any daily or intermittent quality control that is performed, as well as the date(s)performed and the initials of the technologistand reviewing supervisor or director. Additional

Quality

Control

Measures

Additional quality control would include a comparison of immunofluorescencewith isolation in cell culture systems, as well as a correlation of results obtained with the clinical information provided. This form of quality control is readily available to regional laboratories but presentsmore of a problem to smaller laboratories without ready accessto cell cultures. Alterof Quality Frequency and Documentation natives for these latter laboratories would be to Control Performance split specimensor to send their stained slidesto After the initial evaluation of the antiserum a regi.onal laboratory for review until the sensiand conjugates, the issueof the use-day quality tivity and specificity of the test system have

CUMITECH

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VIRUS

been established. After initial verifications the frequency of referral to a regional laboratory would be determined by the number of specimens tested, continued availability of experienced personnel, and comparison of test results with clinical data. Specimen collection Specimen adequacy is of utmost importance to the quality of results, either by isolation or by antigen detection; therefore, quality control of the specimens must be addressed by each laboratory. A specimen with inadequate cells for evaluation (e.g., fewer than 25 ciliated epithelial cells per well in a respiratory sample) should not be reported as negative, but as having inadequate cells for antigen detection. The importance of this type of report increases directly with the reliability of the antigen detection system. It is also imperative that the laboratory communicate directly with the individuals responsible for specimen collection if a trend of inadequate specimens begins to develop. Such specimens are a waste of time, reagents, and money. This problem is usually more easily controlled for specimens collected within the laboratory’s institution than those which are not. One method of addressing this problem with referral specimens is immediate feedback to the requesting physician, both with positive results and with information that the specimen was inadequate. This information has much more impact if given immediately after the initial preparation and evaluation, rather than allowing any delay. The collection of specimens from in-house patients by the technologist has many benefits. First, it educates the technologist on the clinical aspects of the viral syndromes. Second, it provides an opportunity for the education of physicians, nurses, etc., by the technologist. Third, it provides personal identification of the patient by the technologist, who then “goes the extra mile” to provide diagnostic information. This personal attention goes a long way toward providing quality patient care and fostering good physician-laboratory relations. In addition to quality control of the antisera and conjugates and the specimens, attention must also be given to other disposables and reagents used in the procedure. Slides can have or develop a film which allows the cells to wash off during the wash process. This film may be due to the age of the slides or to the manufacturing process. If it appears that specimens are adequate but there are few cells for evaluation at the end of the procedure, precleaning of the slides with acetone or ethanol may be useful.. If cell loss from the slides is a consistent problem, a change of supplier or routine cleaning is indicated to prevent the loss of specimens.

DETECTION

BY IMMUNOFLUORESCENCE

5

The pH of the PBS used for diluting reagents and washing the slides is critical to the success of the procedure. The pH should be between 7.2 and 7.8. The source of the water used for reagents may affect the pH and should be confirmed for each batch of PBS. The pH of the mountant is optimally 8.0 to 9.5. The acetone fixative should be reagent grade. Contamination of the fixative with water may cause a hazy appearance of the cells or result in inadequate fixation (see Table 2). Quality control of the microscope wilI require a record of use-time if a mercury arc source bulb is used. Routine maintenance and cleaning should also be recorded for either type of light source. SELECTION OF SPECIMENS The selection of specimens for antigen detection or isolation is determined by the clinical history and presentation of the patient, local epidemiology, and known natural history of viral infections (5). Knowledge of the existence of local outbreaks is very useful for the conservation of reagents. For example, in the midst of an outbreak of RSV, respiratory specimens should first be tested for RSV antigen and tested for other viruses only if negative or if the patient has a syndrome not typical of RSV. The risk of this approach is that the laboratory may miss a dual infection with a second or third virus. The use of this information in the clinical management of a patient or a research project must be evaluated against the cost of testing all specimens for all possible viral antigens. Likewise, genital lesions generally require testing only for HSV antigen. Lesions from other cutaneous sites, e.g., back and chest, should be tested with antisera for VZV as well as HSV. The clinical history of immunosuppression or immunodeficiency requires the inclusion of HSV and CMV in the differential diagnosis of respiratory infections. Once the clinical information has determined the general category of infections to be considered, the specific specimen(s) must be selected. Various specimens have been used for the diagnosis of respiratory illnesses. Each laboratory or investigator advocates a particular method of specimen collection. Evaluation of these methods must be undertaken carefully. For example, some virologists believe that nasal washes are superior to NP swabs and can provide data to that effect. Before a decision is made that a specific method will not work, it is important to evaluate the precise method of collection for both specimens. The following sections provide a summary of collection methods and potential problems associated with their use for viral antigen detection. NP Swabs NP swabs should be collected with type 1 cotton- or Dacron-tipped aluminum-shafted

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swabs. The swab should be inserted deeply into the nasopharynx and left in place for 45 to 60 s. (NOTE: This is not a nasal swab!) Swabs taken from infants and small children may be smeared directly on wells of slides by carefully rolling all sides of the swab on each well. Three to six wells can usually be made from one swab. A second swab should be taken if viral isolation is also desired. When properly done, this method can yield a large number of antigen-bearing ciliated epithelial cells. The slides are air dried, fixed in acetone, and stained. Throat

Swabs

24

BAL

The diagnosisof the viral etiology of pneumonia in immunocompromised patients is often achieved by using bronchoalveolar lavage (BAL). Both CMV (6, 22) and HSV antigens may often be detected directly in the cells obtained. Although respiratory viruses such as adenovirus, RSV, and influenza virus can be isolated from BAL specimens,the diagnosisof such infections can usually be made by much lessinvasive techniques. Lung Tissue

The presence of viral antigens in lung tissue Swabs of the throat are of limited usefulness may be evaluated by the preparation of impresfor the detection of most respiratory viral antision smearsor frozen sections. If at all possible, gens. They frequently yield large numbers of noninfected lung tissue should be included as a squamousepithelial cells which serve to dilute control in the evaluation of conjugatesif they are the ciliated epithelial cells. Throat swabsare best to be used for this purpose. pooledwith an NP swabfor viral isolation. Pooled NP and throat swabsare especially useful if viCells rusessuch as influenza virus, enteroviruses,adePleural fluid or transthoracic aspirates as a noviruses,HSV, and CMV are suspected. source of cells are of limited value for antigen detection. Often these specimenscontain large Nasal Aspirates Some investigators have found that nasal as- numbers of erythrocytes and leukocytes which pirates are useful specimens.This is especially may nonspecifically bind the antisera or conjutrue if the individuals responsiblefor specimen gates. Care must be exercised in the interpretacollection are trained in the collection method. tion of these smears(2). Clinical history is equally important in the Once received in the laboratory, these speciselection of a specimen(s)for the diagnosis of mens must be centrifuged to pellet the cells, which are then washedfrom one to five times to exanthems and enanthems. remove the excessmucuscollected aspart of the Lesions aspirate. However, each wash required inOne of the most common presentations of an creasesthe potential for loss of cells. exanthem for which viral antigen detection is Oropharyngeal Washes employed is lesions. If the lesion is a vesicle, it Oropharyngeal washesoften fail to yield suf- must be carefully opened to allow gentle but ficient numbersof ciliated epithelial cells for the thorough swabbingof the baseof the lesion. The detection of respiratory viral antigens. Problems epithelial cells provide the most fruitful source are further compoundedby the tendency to use of antigenfor both HSV and VZV (7). The lesion rather large amounts of saline for the wash, should be clean, but not prepared with alcohol which also dilutes the virus for purposes of or iodine before specimen collection; it can be isolation. Repeated washing of the cells is also properly disinfected afterward. Antigen detection may be more sensitive than a culture after required. the lesion has crusted over. Swabsfrom lesions Transtracheal Aspirates may be used to prepare smearsdirectly or may Transtracheal aspiratesare of particular use- be placed in viral transport media. It should be fulness in immunocompromised patients. Al- noted that the sensitivity of antigen detection by though being replaced in some institutions by immunofluorescencefor HSV significantly dispecimensobtained by fiberoptic bronchoscopy minishes when compared with isolation if no or lung biopsy, they are of sufficient quality to lesionsare present; i.e., immunofluorescenceis avoid the problems of nasal aspiratesand oro- exceedingly insensitive in screeningfor the depharyngeal washes. One centrifugation is usu- tection of HSV in cervical or vaginal specimens ally sufficient to pellet and wash the cells for use if no lesionsare present. in direct antigen detection. Transtracheal aspiMacular or Maculopapular Exanthems with or rates may contain more antigen and infectious without Enanthems virus than do NP or throat swabsfor the diagnosis of CMV pneumonia in immunocomproRashesmay be caused by a large number of misedpatients. viruses, particularly adenoviruses, enterovi-

CUMITECH

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VIRUS

ruses, measles, and rubella. Therefore, a pooled NP and throat swab sample is useful for the detection of these antigens. If measles is suspected, the buccal mucosa should be swabbed as well, since the Koplik’s spots often have large numbers of measles antigen-positive cells. The antigen may be particularly well localized to multinucleated epithelial giant cells (12, 21). Conjunctivitis Adenovirus or HSV conjunctivitis may be diagnosed by the collection of an adequate conjunctival swab. The eye discharge is not a useful specimen, and excess discharge should be gently removed before proceeding. The lower eyelid is then everted and the conjunctiva is swabbed carefully but thoroughly. Wire-shafted swabs should not be used for this specimen for safety reasons. The large swab is safer and produces a better specimen. Cornea1 scrapings can also be useful, but should be collected only by a physician who is skilled in the technique. Urine Sediment is useful for the detection of measles and in some cases CMV and (20, mumps, adenovirus antigens (1, 3). The urine must be centrifuged to pellet the cells. If there are a large number of crystals pelleted with the cells, the pellet should be suspended in 12 to 15 ml of distilled water and recentrifuged to avoid fluorescence interference by the crystals. Biopsy and Tissue Specimens Biopsy and tissue specimens may be used for antigen detection by making impression smears or frozen sections. Brain biopsy specimens for the diagnosis of HSV infection may be used to make impression smears before culture. Biopsies of esophagus, stomach, intestine, lung, and myocardium are all acceptable specimens for viral antigen detection. Impression smears are easily made by virology laboratory technologists. The use of slides sterilized by dipping in alcohol and flaming enables the specimens to be further processed for culture after the impression smears are made. Blotting of tissues that are received in viral transport media or saline or that are particularly bloody increases the number of cells that attach to the slide and provides smears that are less likely to exhibit nonspecific binding of the antiserum. Slides are rapidly air dried and then fixed in acetone. Formalin-Fixed, Paraffin-Embedded Tissues Tissues that have been fixed also have been retrieved for immunofluorescent staining in attempts to detect HSV, measles, and rabies (19). Deparaffinized, rehydrated sections are digested with 0.25% trvpsin for 1 h at room temperature,

DETECTION

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7

washed with PBS for 5 min, and then appropriately stained by the direct or the indirect method. TRANSPORT OF SPECIMENS Swabs in transport media (5) should be transported on wet ice if transit requires more than a few minutes. Lavages, washes, and aspirates of the respiratory tract may be transported as collected if the transit time is less than 1 h. Transport medium should be added to the specimen if the transit time is more than 1 h. Tissue and biopsy specimens may be placed in viral transport media or in saline without preservatives for transport. The important concern is to avoid drying of the tissue or biopsy specimen. When smears only are to be referred to the laboratory, the slides may be fixed in acetone at the collection site. If the transport time is less than 12 h, the air-dried slides may be sent without fixation. It should be noted that additional fixation does not adversely affect the specimen and is preferable to processing a specimen that was fixed inadequately or not at all. Slides may be sent at room temperature or cooled. If placed in a package with viral specimens, care should be taken to protect the slides from moisture. LABORATORY

METHODS

Fixatives and Fixation Acetone is the most widely used fixative for specimens containing viral antigens. It should be of reagent grade and free from contamination by water to avoid hazing and to preserve good cellular morphology. Cold acetone (-20 to 8OC) has traditionally been used; however, experience with room temperature acetone has shown that cold fixation is probably more of a tradition than a necessity. The lo-min fixation time has also been traditional. Longer durations do not adversely affect the specimen, and shorter times are probably adequate, as penetration of the cells occurs very quickly. Flooding the slide smear with acetone and allowing it to air dry appear to be adequate. Containers of acetone should be labeled flammable and care should be taken to avoid placing flames near them. Large quantities of acetone should be stored in safety cans. Staining Procedures Consideration must be given to the basic procedure to assure reproducible, quality results. It is also useful in the resolution of technical problems. Incubation times vary from 15 to 60 min. The minimal incubation time to be used in a laboratory is that used when the antisera or coniunates

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are titrated. Increasing the incubation time does not adversely affect the assayas long as drying of the slides is prevented. The sameis true of temperature; both room temperature and 35 to 37OChave been used successfully. Titration of the antisera and conjugatesshouldbe performed at the temperature at which they will be routinely used. Manufacturers of the reagents usually recommendan incubation time and temperature. Wet chambers for incubations can be madeby useof a wet paper towel in a petri plate or plastic chamber. Wooden or plastic applicators or straws may serve to keep the slides elevated from the wet towel. Larger plastic containers (Tupperware type) may be used if many slidesare processedat one time. Laboratories with adequatefunds may wish to purchase commercially available humidified chambers. These, however, require periodic cleaning to avoid growth of fungi. The washstepsare critical and, unfortunately, affected by several factors. In many of the original publications describing immunofluorescenceprocedures, two to five wash stepsof 5 min each in Coplin jars were used. Subsequent recommendationswere for two 5-min washes with gentle agitation. This may be accomplished by the sloshingup and down of the stainingrack in the washing dish by the technologist or by placing the dish on a rotator at 10 to 30 rpm. It shouldbe noted that theserecommendationsare for polyclonal antibodies. The introduction of monoclonal antibodies has required a reevaluation of wash steps.When monoclonal antibodies are used, retention of the traditional longer wash times may yield falsely negative results. Two 1-minwashesare adequatefor most monoclonal antibodies. Alternatives to be explored include a rinse for a few secondsfrom a wash bottle or 10 to 20 dips in PBS. The surface tension of PBS assists greatly in removing excess reagent. When commercial reagentsare used,the recommendations of the manufacturer generally shouldbe followed. Wash procedures shouldbe carefully evaluated and firmly adhered to in order to avoid erroneousresults; also, care must be taken to avoid a low pH of the wash solution, which will inactivate the conjugate dye. Buffered glycerol is the traditional mountantfor immunofluorescentstaining.The pH shouldbe 8.0 to 9.5. Some commercial sourcesof conjugates provide their mounting media, which includes chemicalsto avoid quenching. The addition of thesesubstancesis gearedto their particular system and may causeproblemsif used with other reagents.If a green hazing appearson the slides, the mountingmedium,as well as the fixation and washingprocedures, shouldbe consideredwhen the problem is evaluated.

24

CUMITECH

TABLE

1. Immunofluorescent antigens Staining pattern

Virus Nuclear

Adenovirus Coronavirus CMV Enteroviruses HSV Influenza Measles Mumps Parainfluenza Rabies RSV vzv

+ + +

Cytoplasmic

+ + t + +

+ + + + + + t

localization

of

Usual cell type expressing antigen”

CRE, Ep CRE EP, 0

0

Ep, Neur, 0 CRE CRC

EP,

Neur, 0 CRE, Ep CRE Neur CRE Ep, Neur

“Abbreviations: CRE, ciliated respiratory epithelial; Ep, epithelial; Neur, neurons; 0, other affected cells.

Localization of Antigen in Positive Specimens The cellular sites of antigen expression vary according to the virus sought, and the specific localization aids significantly in determining the true specificity of the test (25). Expected staining patterns for various viruses are summarizedin Table 1. When the localization criteria for positivity are not met, one mustaddressthe possibility of nonspecificstaining,describedbelow. Technical Problems Technical problemswith immunofluorescence assays are usually relatively easy to resolve. Potential problems and considerations for their resolution are listed in Table 2. Nonspecific staining of clinical specimens may result from a variety of sources.Technical problems include inaccurate dilution of antisera or conjugates, inadequate washing procedures, and allowing the slides to dry during incubations. The specimensmay also be the source of this problem. Leukocytes may bind the conjugate nonspecifically; such fluorescence is usually membrane associated and more homogeneous than granular. Careful focusing of the microscope will often reveal the multilobed nucleus of the polymorphonuclear cell. Erythrocytes will also bind someconjugates. The size, disk shape, and smooth homogeneousappearance of these cells should not confuse the laboratorian. Yeasts in specimens may autofluoresee, and these or bacterial contaminants such as Staphylococcus aureus nonspecifically bind antibodies from some sources. Respiratory specimensfrom infants who have been recently fed formula may bind the conjugate to produce an overall, low-level green glow. Offering the infant water before specimen collection or

TABLE

VIRUS DETECTION

24

CUMITECH

BY IMMUNOFLUORESCENCE

9

2. Technical problems often encountered

Problem

Possible reasons

Too few cells

Inadequate specimen collection; improper fixation; too vigorous washing; slides need to be cleaned

False-negative results

Inaccurate dilution of antisera or conjugates; wrong pH of diluents, wash solutions, or mounting media; excessive washing times, especially with some monoclonal reagents; antibody no longer reactive; FITC label disassociated from antibody; wrong FITC-labeled antisera in indirect system

Nonspecific fluorescence

Inadequate washing; improper titration or dilution of antisera or conjugates; drying of slides during incubations; specimen related, e.g., mucus

avoiding feedingtime will alleviate this problem. Mucus on the cells will bind the conjugate as well. Theseproblemswith direct specimensmay be significant to the novice, but false-positives should not result if strict criteria for staining patterns are observed. Most viral antigensAuoresee in granular patterns, while nonspecific fluorescenceusually appearssmoothand homogeneous,often highlighting the entire cell population being examined. It should also be noted that the staining patterns with monoclonal antibodies may vary with their epitope specificity and can differ significantly from the patterns observedwith polyclonal antisera. The possibility of cross-reactivity of the viral antiserumwith tissue antigens, especially HLA antigens, must also be considered. This can occur with monoclonal as well as polyclonal antisera. When expected staining patterns are observed in appropriate cells and only a portion of all appropriate cell types exhibit fluorescence,nonspecificity becomesmuch lesslikely. Shell Vial Assay Methods An alternative to the detection of antigensin cellsor tissuesobtained directly from patients is the amplification of the virus in cell cultures for a short time, with subsequentimmunofluorescencestainingof the infected monolayersfor the detection of early viral antigens. By this procedure a low titer of virus present in a specimen can replicate in cell cultures, thereby amplifying the virus or early antigens and number of infected cells compared with the original specimen. For the herpesvirusesin particular, this increase in the quantity of antigen is generally reflected by a greater sensitivity of a fluores-

FIG. 2. Diagram of shell vials with medium (left) and cover slip with cell monolayer (right).

cence assaycomparedwith the direct detection of virus-infected cells obtainedfrom patients. In addition, debris consisting of broken cellular fragments and other background material in a specimenextract or scrapingsthat can be detrimental to the specific interpretation of the fluorescencereaction is substantially reduced after inoculation into shell vial cell cultures. In the past, substantial efforts were made to develop rapid assaysfor the detection of CMV sincethis virus usually requiresa meanof 8 to 10 days before it can be recognized by cytopathic effect in conventional tube cell cultures. Stagno et al. were the first to develop a fluorescence assayfor CMV, using a human source of antisera or hyperimmune sera prepared in guinea pigs to detect nuclear antigens of CMV in a microculture system 24 h after inoculation with the specimen(33). Similarly, Goldstein et al. and Volpi et al., using monoclonal antibodies specific for CMV, detected fluorescent foci produced by the virus in open lung biopsies or tissue specimens(16, 36). The shell vial assay was initially developed for the rapid detection of CMV (14), basedon the studiescited above, the availability of a monoclonalantibody specificfor an early antigen of CMV, and existing technology for the diagnosisof Chlamydia trachomatis in cell cultures. General descriptionof procedure Specimensare inoculated into l-dram shell vials containing 12-mmround cover slipswith a monolayer of appropriately susceptible cells (Fig. 2). After inoculation the shell vials are closedwith rubber stoppers,placedin centrifuge carriers that can accommodateseveral vessels, and centrifuged at 700 x g for 40 min (model RT6000 centrifuge, Du Pont Co., Wilmington, Del). After the centrifugation step, 1 ml of medium is added to each vial and the cultures are incubated at 36°C for 16 to 24 h. After incubation the mediumis aspiratedand the vials

10

MINNICH

ET AL.

are filled with cold acetone. After a lo-min fixation of the cell monolayers, the acetone is aspirated and the reagent is allowed to evaporate completely. Cover slips with monolayers are then stained with monoclonal antibodies preferentially directed to immediate early antigens of the virus and then subsequently reacted with a fluorescein-conjugated goat anti-mouse immunoglobulin G in an indirect immunofluorescence test. The cover slips are them removed from the vials, blotted on tissue paper, and mounted (cell side down) using mounting fluid. Procedure variables for the detection of CMV The shell vial assay consists of several steps that must be performed in a uniform manner to produce results that are sensitive and accurate. A number of variables have been identified, particularly regarding the early detection of CMV from a variety of clinical sources. (i) Cell cultures. Cover slips in shell vials are seeded with a cell suspension to produce a monolayer within 1 to 3 days. Cultures should be inoculated with specimens within a week after preparation. Older cell monolayers tend to be more sensitive to the toxic effects of some specimens than are fresh cultures. Confluent monolayers of cells on cover slips are required for the maximum detection of CMV. Although simultaneous seeding of shell vials with cells and the inoculum would eliminate planning for advance monolayer cultures, the detection of CMV is significantly reduced in this system. For example, of 50 isolates that were detected by immunofluorescence in shell vials containing confluent monolayers, only 39 (78%) were detected in shell vials that had been seeded and inoculated simultaneously (P < 0.001) (8, 10). Treatment of MRC-5 cell monolayers with dimethyl sulfoxide-dexamethasone or iododeoxyuridine solutions 24 h before inoculating the cultures with specimens containing CMV does not appear to increase the sensitivity for detecting the virus compared with untreated controls. Two shell vials are inoculated with all specimens except for blood (three shell vials). A retrospective evaluation of 83 blood culture specimens positive for CMV indicated that inoculation of three shell vials increased the rate of detection of the virus by 51% compared with the use of one vial. Inoculation of three shell vials compared with two yielded a 20% increase in the detection of CMV. For urine, tissue, and BAL, inoculation of two shell vials compared with one resulted in increases of less than 10%. (ii) Specimens. Urine, BAL, and the supernatant fraction of tissue extracts are inoculated directly into shell vials. Mononuclear and polymorphonuclear cells of blood specimens separated by barrier gradients (e.g., FicollPaque and

CUMITECH

24

Macrodex) are inoculated into three shell vials. Other specimens (swabs of dermal or respiratory sites) are extracted into 2 ml of serum-free, antibiotic-containing medium and then inoculated into shell vials. Inocula of 0.3 to 0.5 ml produce more fluorescent foci than do lower volumes; however, the background debris can cause nonspecific fluorescence and higher volumes of specimen material can cause toxicityproducing morphologic changes in host cells. An inoculum of 0.2 ml provides a reasonable balance between optimal sensitivity of the assay and interfering background of debris that can be a problem with the immunofluorescence shell vial assay when higher volumes are used. . Specimen toxicity, even with an inoculum volume of 0.2 ml, is generally associated with shell vials that are more than 1 week old; however, specimens such as blood or BAL that are composed of cellular material produce host cell toxicity more frequently than do urine specimens. Conventional tube cell cultures are more likely than shell vials to develop toxicity associated with the specimen because of the prolonged contact of the inoculum material with the monolayers. This is most frequently observed for urine specimens. (iii) Centrifugation. Centrifugation (spin amplification) is probably the single most important step in the shell vial assay. Low-speed centrifugation (700 x g) of the specimen inoculum onto monolayers has been shown to increase the infectivity of several microorganisms, such as Chlamydia spp. (3l), murine and human strains of CMV, HSV (35), adenovirus, and rotavirus (4). Two of the earliest studiesindicated a 20- to 80-fold increase in the replication of murine CMV in mouseembryo cultures (18, 27). Similarly, low-speedcentrifugation of clinical strains of CMV produced 3- to 14-fold increasesin the number of fluorescent foci detected in shellvials 16 h postinoculation. Initial studies indicated that only 6 of 16 (38%) clinical strains of CMV were detected without the spin amplification step (14). With centrifugation the shell vial assay is probably more sensitive than conventional cell culture in tubes for the detection of CMV from all specimens,especially urine (15, 28). Of 109 CMV strains from urine detected in shell vials, only 77 were recovered in the noncentrifuged standardtube cell cultures. The specificity of the shell vial assay was indicated by the recovery of the virus from urine specimensand the demonstration of increased antibody to the virus in sera collected from these patients (15). In other studies that have used monoclonal antibodies for the detection of early antigens of CMV but have not used the centrifugation step, the sensitivity of the rapid assaywas only 80% comparedwith the recovery of CMV in conven-

CUMITECH

24

VIRUS

DETECTION

tional tube cell cultures (17, 34). Spin amplification also enhances the detection of clinical HSV strains compared with cell cultures that were not centrifuged (13, 24, 32; T. F. Smith, M. J. Espy, D. J. Wilson, and A. D. Wold, Clin. Immunol. Newsl. 8:43-46, 1987). Centrifugation at 700 x g is adequate. Higher forces (1,500 x g) frequently damagethe monolayers, as indicated by cellular distortions and lysis that are evident microscopically. Becausea gravitational force of 700 X g (2,000 rpm) is easily obtained with most tabletop centrifuges, most clinical laboratories can perform this key step in the shell vial assay without investing in specialequipment. Approximately two- to threefold-higher numbers of fluorescent foci are obtained by centrifugation at 36 compared with 25°C(Smith et al., Clin. Immunol. Newsl. 8:4346, 1987). (iv) Incubation. Even though infected cells can be detected by fluorescenceas early as2 h after inoculation with laboratory-passagedCMV and 4 to 6 h with clinical strains of CMV, the minimum amount of time required for the consistent detection of the early antigen of the virus is approximately 16h. Most laboratories receive specimensin the late morning and inoculate them into shell vials in the afternoon, and the results of an assay at 4 to 6 h postinoculation would be available only after normal working hours. Thus, an assayat 6 h postinfection probably would not be significantly more advantageous than an assay performed at 16 h postinfection. The formation and accumulation of the early antigen of CMV are not enhancedby incubating shellvial cell cultures at higher temperatures(39 to 42 versus 36°C) (Smith et al., Clin. Immunol. Newsl. 8:43-46, 1987). (v) Staining of cover slips. The indirect fluorescent antibody method is most commonly used for the detection of CMV antigens. However, a commercially available reagent for use in the direct test has been shown to be as sensitive as the indirect procedure for the detection of CMV from clinical specimens(11). The staining procedure can be carried out by addingthe reagents directly to the monolayer in the shell vial. The choice of fluoresceinated anti-mouse conjugate seemsto be relatively unimportant regarding specificity for the H- or L-chain, Fc or F(ab), componentsof the immunoglobulin G antibody molecule. Stained cover slips are removed from shell vials by using a needle with a bent tip attached to one arm of a forceps. The cover slips are blotted on absorbenttissueto remove excess moisture and then placed on a glass slide (cell sidedown) with a drop of mounting medium(pH 8.0 to 9.5). Alternatively, square cover slips (9 by 9 mm) may be usedin standard2-dram vials.

BY IMMUNOFLUORESCENCE

11

These offer the advantagesthat both the rubber or Teflon-lined caps and glass vials may be sterilized by autoclaving and thesevials easily fit in the standard centrifuge carrier used for test tubes that are 13 to 16 by 100mm. In addition, the cells can be fixed with acetone in the vials and then the cover slips can be removed for further staining. This can conserve the amount of potentially expensive reagentsused. After the cells have been fixed with acetone, it is important that the cover slips are allowed to dry completely. Residualacetone can produce trapping of the monoclonal antibody and therefore causenonspecific staining. (vi) Examination of stained cover slips. The 2H2.4 monoclonal antibody detects an early 72,000-dalton antigen of CMV that homogeneously fills the nuclei of infected cells 16 h postinoculation. The regular shape of the stained infected nuclei allows for the specific detection of virus-infected cells and easily differentiates these cells from nonspecific staining of broken cells and other debris (Fig. 3A). Detection

of other viruses by shell vial assay

The successof rapid detection of virus infections by the shell vial assay depends on the availability of monoclonalantibodies directed to immediate early antigens of the virus. Accordingly, application of the shell vial method has been reported for the detection of HSV, VZV, influenza virus, and adenovirus. Although most HSV strains can be detected in conventional tube cell cultures 1 to 3 days postinoculation, generally 5 to 7 days is required for maximum sensitivity of this system. With monoclonal antibodies specific for HSV types 1 and 2, two shell vials are inoculated with genital specimensand then each cell culture is stained with the specific antiserum 16 h after inoculation. If the specimencontains HSV, only one of the two cover slipswill contain fluorescent cells in plaquelike foci typical for HSV (Fig. 3B). The other stained cover slip serves as a control and should not contain fluorescent foci. Polyclonal antisera, pooled monoclonal antibodies to HSV types 1 and 2, and a monoclonal antibody to HSV group antigens may also be used with the shell vial assay for HSV. Dermal specimens contain either HSV or VZV, unlike those from most genital sources which do not need to be evaluated for VZV. Therefore, four shell vials are inoculated with the specimenextract. Cover slipsfrom two shell vials are stained with monoclonal antibodies specific for the serotypes of HSV 16 h postinoculation, and the remaining monolayers on the shell vial cover slip are reacted with a monoclonal antibody to VZV after 2 days and, if nega-

12

MINNICH

FIG. 3. Monolayers (C), adenovirus (D),

ET AL.

on cover slips stained with monoclonal influenza virus type A (E), and influenza

tive, at 5 days of incubation (Fig. 3C) (Smith et al., Clin. Immunol. Newsl. 8:43-46, 1987). The shell vial assay for the detection of adenovirus has been most useful with specimens from the eye. Four shell vials are inoculated, two are stained with monoclonal antibodies to HSV and two are stained with a monoclonal antibody that reacts with the group antigen of adenovirus at 3 and 5 days postinoculation (Fig. 3D) (9). Lastly, the shell vial technique can be usedto rapidly detect influenza virus infection and to conveniently differentiate the agent according to serotype A or B (Fig. 3E and F, respectively) (11). SUMMARY

Immunofluorescenceis exceedingly useful for the rapid, direct detection of viral antigens in clinical specimens,particularly if the number of antigensto be considered is small and adequate antigen-bearing cells are present. When the amount of antigen in a specimenis insufficient for direct detection, amplification in a cell culture by use of the shell vial assay can enhance detection. The successof these methods is dependent upon the appropriate selection and collection of specimens,careful quality control of reagents,standardization of techniques, and the knowledgeand skill of the individual performing the procedure. When appropriately used, along with good physician-laboratory communication, immunofluorescence offers a valuable adjunct to isolation systems and provides a method of rapid, accurate diagnosisof viral infections.

24

CUMITECH

antibodies virus type

reactive B (F).

to CMV

LITERATURE

(A),

HSV

(B),

VZ

CITED

1. Alpert, G., M.-C. Mazeron, R. Coliion, and S. Plotkln. 1985. Rapid detection of human cytomegalovirus in urine of humans. J. Infect. Dis. 152631-633. 2. Bartlett,

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T. F. Smith,

and W. R. Wilson.

1987. Cumitech 7A, Laboratory diagnosis of lower respiratory tract infections. Coordinating ed., J. A. Washington II. American Society for Microbiology, Washington, D.C. 3. Belsle, R. B., and M. A. M&on. 1974. Identification by immunofluorescence of adenoviral antigen in exfoliated bladder epithelial cells from patients with acute hemorrhagic cystitis. hoc. Sot. Exp. Biol. Med. 146:754-758. 4. Cevenini, R., F. Rumpianesi, E. Falcieri, and I. Sarov.

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1987. Evaluation of three assays on alveolar lavage fluid in the diagnosis of cytomegalovirus pneumonitis after bone marrow transplantation. J. Infect. Dis. 155:495-500. 7. Drew, W. L., and L. Mintz. 1980. Rapid diagnosis of varicella-zoster virus infection by direct immunofluorescence. Am. J. Clin. Pathol. 73699-701. 8. Emanuel, D., J. Peppard, strong, and U. Hammerling.

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1986. Rapid immunodiagnosis of cytomegalovirus pneumonia by bronchoalveolar lavage using human and murine monoclonal antibodies. Ann. Intern. Med. 104:476-481. 9. Espy, M. J., J. C. Hierholzcr, and T. F. Smith. 1987. The effect of centrifugation on the rapid detection of adenovirus in shell vials. Am. J. Clin. Pathol. 88:358-360. 10. Espy, M. J., and T. F. Smith. 1987. Simultaneous seeding and infecting of shell vials for rapid detection of cytomegalovims infection. J. Clin. Microbial. 25:940-941. 11. Espy, M. J., T. F. Smith, M. W. Harmon, and A. P. Kendal. 1986. Rapid detection of influenza virus by shell vial assay with monoclonal antibodies. J. Clin. Microbial. 24:677-679. 12. Fulton, R. E., and P. J. Middleton. 1975. Immunofluores-

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VIRUS

cence in diagnosis of measles infections in children. J. Pediatr . 86: 17-22. Gleaves, C. A., C. F. Lee, L. Kirsch, and J. D. Meyers. 1987. Evaluation of a direct fluorescein-conjugated monoclonal antibody for detection of cytomegalovirus in centrifugation culture. J. Clin. Microbial. 25: 1548-l 550. Gleaves, C. A., T. F. Smith, E. A. Shuster, and G. R. Pearson. 1984. Rapid detection of cytomegalovirus in MRC-5 cells inoculated with urine specimens by using low-speed centrifugation and monoclonal antibody to an early antigen. J. Clin. Microbial. 19:917-919. Gleaves, C. A., T. F. Smith, E. A. Shuster, and G. R. Pearson. 1985. Comparison of standard tube and shell vial cell culture techniques for detection of cytomegalovirus in clinical specimens, J. Clin. Microbial. 21:217-222. Goldstein, L. C., J. McDougall, R. Hackman, J. D. Meyers, E. D. Thomas, and R. C. Nowinski. 1982. Monoclonal antibodies to cytomegalovirus: rapid identification of clinical isolates and preliminary use in diagnosis of cytomegalovirus pneumonia. Infect. Immun. 38:273-28 1. Griffiths, P. D., D. D. Panjwani, P. R. Sitrk, M. G. Ball, M. Ganczakowski, H. A. Blacklock, and H. G. Prentice. 1984. Rapid diagnosis of cytomegalovirus infection in immunocompromised patients by detection of early antigen fluorescent foci. Lancet ii: 1242-1244. Hudson, J. B., V. Misra, and T. R. Mosman. 1976. Cytomegalovirus infectivity: analysis of the phenomenon of antifungal enhancement of infectivity. Virology 72:235243. Johnson, K. P., P. T. Swoveland, and R. W. Emmons. 1980. Diagnosis of rabies by immunofluorescence in trypsin-treated histologic section. J. Am. Med. Assoc. 244:4143. Kao, C. L., K. McIntosh, B. Fernie, A. Talia, L. Pierik, and L. Anderson. 1984. Monoclonal antibodies for the rapid diagnosis of respiratory syncytial virus infection by immunofluorescence. Diagn. Microbial. Infect. Dis. 2: 199-206. Llanes-Rodas, R., and C. Liu. 1966. Rapid diagnosis of measles from urinary sediments stained with fluorescent antibody. N. Engl. J. Med. 275:516-523. Martin, W. J., II, and T. F. Smith. 1986. Rapid detection of cytomegalovirus in bronchoalveolar lavage specimens by a monoclonal antibody method. J. Clin. Microbial. 23: 1005-1008. McQuillan, J., C. R. Madeley, and A. P. Kendal. 1985. Monoclonal antibodies for the rapid diagnosis of influenza A and B virus infections by immunofluorescence. Lancet ii:91 1-914. Michalski, F. J., M. Shaikh, F. Sahraie, S. Desai, L. Verano. and J. Vallabhanei. 1986. Enzvme-linked im-

DETECTION

25.

26.

27. 28.

29.

30. 3 1. 32.

33.

34.

35. 36.

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BY IMMUNOFLUORESCENCE

13

munosorbent assay spin amplification technique for herpes simplex virus antigen detection. J. Clin. Microbial. 24:310-311. Minnich, L. L., and C. G. Ray. 1980. Comparison of direct immunofluorescent staining of clinical specimens for respiratory virus antigens with conventional isolation techniques. J. Clin. Microbial. 12:391-394. Minnich, L. L., 2. M. Shehab, and C. G. Ray. 1987. Application of pooled monoclonal antibodies for one-hour detection of respiratory syncytial virus antigen in clinical specimens. Diagn. Microbial. Infect. Dis. 7: 137-l 4 1. Osborn, J. E., and D:L. Walker. 1968. Enhancement of infectivity of murine cytomegalovirus in vitro by centrifugal inoculation. J. Virol. 2:853-858. Paya, C. V., A. D. Wold, and T. F. Smith. 1987. Detection of cytomegalovirus in specimens other than urine by the shell vial assay and conventional tube cell cultures. J. Clin. Microbial. 25:755-757. Pothier, P., J. C. Nicolas, G. P. de Saint Maur, S. Ghim, A. Kazmierczak, and F. Bricout. 1985. Monoclonal antibodies against respiratory syncytial virus and their use for rapid detection of virus in nasopharyngeal secretions. J. Clin. Microbial. 21:286-287. Ray, C. G., and L. L. Minnich. 1987. Efficiency of immunofluorescence for rapid detection of common respiratory viruses. J. Clin. Microbial. 25:355-357. Reeve, P., J. Owen, and J. D. Oriel. 1975. Laboratory procedures for the isolation of Chlamydia trachomatis from the human genital tract. J. Clin. Pathol. 28:910-914. Salmon, V. C., R. B. Turner, J. J. Speranza, and J. C. Overall, Jr. 1986. Rapid detection of herpes simplex virus in clinical specimens by centrifugation and immunoperoxidase staining. J. Clin. Microbial. 23:683-686. Stagno, S., R. F. Pass, D. W. Reynolds, M. Moore, A. J. Nahmias, and C. A. Alford. 1980. Comparative study of diagnostic procedures for congenital cytomegalovirus infection. Pediatrics 65:25 l-257. Swenson, P. D., and M. K. Kaplan. 1985. Rapid detection of cytomegalovirus in cell culture by indirect immunoperoxidase staining with monoclonal antibody to an early nuclear antigen. J. Clin. Microbial. 21:669-673. Tenser, R. B., and M. E. Dunstan. 1980. Mechanisms of herpes simplex virus infectivity enhanced by ultracentrifugal inoculation. Infect. Immun. 30:193-197. Volpi, A., R. J. Whitley, R. Ceballos, S. Stagno, and L. Pereira. 1983. Rapid diagnosis of pneumonia due to cytomegalovirus with specific monoclonal antibodies. J. Infect. Dis. 147:1119-l 120. Waner, J. L., N. J. Whitehurst, T. Downs, and D. G. Graves. 1985. Production of monoclonal antibodies against parainfluenza 3 virus and their use in diagnosis by immunofluorescence. J. Clin. Microbial. 22:535-538.